Detection of 5T4 RNA in plasma and serum

ABSTRACT

This invention provides methods for detecting the presence of malignant or premalignant cells, or trophoblastic cells in a human wherein the malignant, premalignant or trophoblastic cells express 5T4. The methods of the invention detect 5T4 RNA in blood, blood plasma, serum, and other bodily fluids. The inventive methods are useful for detection, diagnosis, monitoring, treatment, or evaluation of neoplastic disease, and for the detection and evaluation of placental tissue in pregnant women.

This application is a continuation of U.S. patent application Ser. No.10/363,023, filed on Jul. 17, 2003, now U.S. Pat. No. 7,767,422, grantedon Aug. 3, 2010, which is a U.S. National Stage application ofInternational Patent Application No. PCT/US01/26119, filed on Aug. 21,2001, which claims priority of U.S. patent application Ser. No.09/649,371, filed on Aug. 28, 2000, now U.S. Pat. No. 6,794,135, grantedon Sep. 21, 2004

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to methods for detecting ribonucleic acid (RNA)in bodily fluids such as blood plasma and serum obtained from an animal.Specifically, the invention is directed towards methods for detectingRNA in bodily fluids from a human bearing a premalignant lesion or amalignancy, ranging from localized neoplasia to metastatic disease. Themethods of the invention are particularly drawn to detecting mRNAencoding all or a portion of a particular gene, referred to herein asthe 5T4 gene. This gene is expressed in many malignant and premalignanttissues, as well as in placental tissue. Since RNA is essential forexpressing the 5T4 gene and producing 5T4 protein, detection andmonitoring of 5T4 mRNA provides a convenient and reliable method forassessing and monitoring 5T4 gene expression.

2. Background of the Related Art

Pathogenesis and regulation of cancer is dependent upon the translationof RNA to produce proteins involved with a variety of cellularprocesses, such as cell proliferation, regulation, and death. ParticularRNAs involved in these cellular processes include 5T4 RNA; these RNAsare associated with cellular processes characteristic of cancer, such asmetastatic potential, invasiveness, and alterations of cell-cellinteractions. Furthermore, some RNA and their translated proteins,although not necessarily involved in specific neoplastic pathogenesis orregulation, may serve to delineate recognizable characteristics ofparticular neoplasms by either being elevated or inappropriatelyexpressed.

The 5T4 protein encoded by said RNA is a transmembrane glycoproteinnormally present in trophoblast tissue whose gene structure has recentlybeen characterized (Hole & Stern, 1988, Br. J. Cancer 57: 239-246; Hole& Stern, 1990, Int. J. Cancer 45: 179-184; Myers, 1994, J. Biol. Chem.269: 9319-9324; King et al., 1999, Biochimica et Biophysica Acta 1445:257-270). The protein is expressed at low levels in cells of only a fewother normal epithelia. Significantly, 5T4 expression is upregulated incells comprising many cancers and premalignant tissues, including butnot limited to cancers of breast, ovary, lung, cervix, colorectum,stomach, pancreas, bladder, endometrium, brain, kidney, and esophagus(Jones et al., 1990, Br. J. Cancer 61: 96-100; Southall et al., 1990,Br. J. Cancer 61: 89-95; Starzynska et al., 1992, Br. J. Cancer 66:867-869; Starzynska et al., 1994, Br. J. Cancer 69: 899-902). Because ofthis, 5T4 mRNA is considered herein to be a tumor-associated RNA.Overexpression of 5T4 is particularly associated with cancers of highmetastatic potential and poorer prognosis (Mulder et al., 1997, Clin.Cancer Res. 3: 1923-1930; Starzynska et al., 1994, ibid.). Detection of5T4 thereby permits detection and monitoring of a wide spectrum ofcancers and premalignancies, and may have prognostic significance. 5T4further provides a target for cancer therapies, particularly monoclonalantibody-based therapies and vaccine therapies.

RNAs associated with cancer and premalignancy have been characterized astumor-derived, and are termed “tumor-associated RNA” herein. Co-ownedand co-pending U.S. patent application Ser. No. 09/649,371, incorporatedby reference herein in its entirety, provides methods for detectingtumor-associated or tumor-derived 5T4 RNA in bodily fluids such as bloodplasma and serum, wherein said RNA detection is used for the detection,monitoring, or evaluation of cancer or premalignant conditions.

Another biological phenomenon in which RNA and translated proteinstherefrom are fundamentally involved is the cascade of events that occurduring pregnancy. Such RNAs assist in the rapid growth of specifictissues during pregnancy, being principally those of the embryo andthose of the placenta, wherein placental tissue includes fetaltrophoblast tissue. 5T4 is normally present in fetal trophoblast tissue,so that detection of 5T4 RNA in bodily fluids such as blood, includingblood plasma and serum, from pregnant or recently pregnant women,enables the monitoring of placental conditions and states in thesewomen, both normal and pathologic. Examples where such monitoring wouldbe advantageous include but are not limited to monitoring of placentalgrowth during pregnancy, evaluation of deleterious conditions associatedwith pregnancy such as preeclampsia and eclampsia, evaluation forretained placenta following normal delivery or following spontaneous orincomplete abortions or medical terminations of pregnancy, andmonitoring of gestational trophoblastic diseases. In contradistinctionwith the invention disclosed herein, which enables detection of RNAspecific for the trophoblastic (placental) compartment of conceptionrather than the entire fetal compartment (including the embryoniccompartment), the presence of fetal RNA in blood plasma has recentlybeen described (Poon et al., 2001, Clin. Chem. 47: 363, Abst. No. 11).

5T4 RNA being recognized herein as a tumor-associated RNA, there is aheretofore unappreciated need in the art to identify premalignant ormalignant states characterized by 5T4 in a human by detecting 5T4 RNA inbodily fluids such as blood plasma or serum. Similarly, since 5T4 is anormal trophoblastic protein, a blood test for 5T4 mRNA in pregnant orrecently pregnant women without cancer would be useful for diagnosingcomplications of pregnancy.

SUMMARY OF THE INVENTION

The present invention provides methods for evaluating a non-pregnantanimal, most preferably a human, for premalignant or malignant states,disorders, or conditions, and further describes methods for evaluating apregnant or recently pregnant woman for placental disorders orconditions, including the existence of the pregnancy per se. Theinventive methods comprise detecting 5T4 RNA in bodily fluids,preferably blood and most preferably blood plasma and serum, as well asin other bodily fluids, preferably urine, effusions, ascites, amnioticfluid, saliva, cerebrospinal fluid, cervical, vaginal, and endometrialsecretions, gastrointestinal secretions, bronchial secretions, breastfluid, and associated tissue washings and lavages.

In preferred embodiments, the methods of the invention comprise the stepof amplifying and detecting extracellular 5T4 RNA from bodily fluids ofan animal, most preferably a human.

In particularly preferred embodiments, the present invention providesmethods for detecting 5T4 RNA in blood or a blood fraction, includingplasma and serum, and other bodily fluids, the method comprising thesteps of extracting RNA from blood, plasma, serum, or other bodilyfluid, in vitro amplifying 5T4 mRNA or its cDNA, and detecting theamplified product of 5T4 mRNA or its cDNA.

In a first aspect of this embodiment, the present invention providesmethods for detecting 5T4 RNA in blood or blood fractions, includingplasma and serum, in an animal, most preferably a human. Said methodsare advantageously provided for detecting, diagnosing, monitoring orevaluating proliferative disorders, particularly stages of neoplasticdisease, including premalignancy, early cancer, non-invasive cancer,carcinoma in-situ, invasive cancer, metastatic cancer and advancedcancer, as well as benign neoplasms. In this aspect, the methodcomprises the steps of extracting RNA from blood or blood plasma orserum, in vitro amplifying qualitatively or quantitatively a fraction ofthe extracted RNA or the corresponding cDNA wherein said fractioncomprises 5T4 RNA, and detecting the amplified product of 5T4 RNA or itscDNA.

The invention in a second aspect provides methods for detecting 5T4 RNAin any bodily fluid. Preferably, said bodily fluid is whole blood, bloodplasma, serum, urine, effusions, ascitic fluid, amniotic fluid, saliva,cerebrospinal fluid, cervical secretions, vaginal secretions endometrialsecretions, gastrointestinal secretions, bronchial secretions includingsputum, secretions or washings from the breast, and other associatedtissue washings from an animal, most preferably a human. In this aspect,the method comprises the steps of extracting RNA from the bodily fluid,in vitro amplifying 5T4 RNA comprising a fraction of the extracted RNAor preferably the corresponding cDNA into which the RNA is converted ina qualitative or quantitative fashion, and detecting the amplifiedproduct of 5T4 RNA or cDNA.

In these embodiments, the inventive methods are particularlyadvantageous for detecting, diagnosing, monitoring, or evaluatingvarious proliferative disorders in a non-pregnant animal, mostpreferably a human, particularly stages of neoplastic disease, includingpremalignancy, early cancer, non-invasive cancer, carcinoma-in-situ,invasive cancer, metastatic cancer and advanced cancer as well as benignneoplasm.

The invention also provides methods for detecting, diagnosing,monitoring, or evaluating pregnancy and placental disorders andconditions in a woman having a current or antecedent pregnancy,particularly disorders and conditions characterized by or associatedwith trophoblast tissue including placental insufficiency, preeclampsia,eclampsia, gestational trophoblastic diseases such as molar pregnancy,gestational trophoblastic neoplasia, and conditions associated withinappropriately retained trophoblastic tissue, such as following laborand delivery, and following recognized or unrecognized earlyterminations of the pregnancy, such as in spontaneous or incompleteabortions or following medical termination of the pregnancy.

The methods of the invention are additionally useful for identifying 5T4RNA-expressing cells or tissue in an animal, most preferably a human. Inthese embodiments, detection of an in vitro amplified product of 5T4 RNAusing the methods of the invention indicates the existence of5T4-expressing cells or tissue in a human.

The invention advantageously provides oligonucleotide primers useful inthe efficient amplification of 5T4 mRNA or cDNA from bodily fluid, mostpreferably blood plasma or serum.

The invention further provides diagnostic kits for detecting 5T4 RNA inbodily fluid, preferably blood plasma or serum, wherein the kitcomprises oligonucleotide primers, probes, or both primers and probesfor amplifying and/or detecting 5T4 RNA or cDNA derived therefrom.

In preferred embodiments of the inventive methods, 5T4 RNA is extractedfrom whole blood, blood plasma or serum, or other bodily fluids using anextraction method such as gelatin extraction method; silica, glass bead,or diatom extraction method; guanidinium thiocyanate acid-phenol basedextraction methods; guanidinium thiocyanate acid based extractionmethods; methods using centrifugation through cesium chloride or similargradients; phenol-chloroform based extraction methods; or othercommercially available RNA extraction methods. Extraction mayalternatively be performed using one or a multiplicity of probes thatspecifically hybridize to 5T4 RNA.

In preferred embodiments of the inventive methods, 5T4 RNA or cDNAderived therefrom is amplified using an in vitro amplification methodsuch as reverse transcriptase polymerase chain reaction (RT-PCR); ligasechain reaction; DNA signal amplification; amplifiable RNA reporters;Q-beta replication; transcription-based amplification; isothermalnucleic acid sequence based amplification; self-sustained sequencereplication assays; boomerang DNA amplification; strand displacementactivation; cycling probe technology; or any combination or variationthereof.

In preferred embodiments of the inventive methods, detecting anamplification product of 5T4 RNA or 5T4 cDNA is accomplished using adetection method such as gel electrophoresis; capillary electrophoresis;conventional enzyme-linked immunosorbent assay (ELISA) or modificationsthereof, such as amplification using biotinylated or otherwise modifiedprimers; nucleic acid hybridization using specific labeled probes, suchas fluorescent-, radioisotope-, or chromogenically-labeled probe;Southern blot analysis; Northern blot analysis;electrochemiluminescence; laser-induced fluorescence; reverse dot blotdetection; and high-performance liquid chromatography.

In particularly preferred embodiments of the inventive methods, 5T4 RNAis converted to cDNA using reverse transcriptase after extraction of RNAfrom a bodily fluid and prior to amplification.

The methods of the invention are advantageously used for providing adiagnosis or prognosis of, or as a predictive indicator for determiningrisk for a human for developing a proliferative, premalignant,neoplastic, or malignant disease comprising or characterized by theexistence of cells expressing 5T4 RNA.

The methods of the invention are particularly useful for providing adiagnosis of or for identifying in non-pregnant animals, particularlyhumans, individuals at risk for developing or who have developedmalignancy or premalignancy of the cells comprising epithelial tissues.Most preferably, malignant or premalignant diseases, conditions, ordisorders advantageously detected or diagnosed using the methods of theinvention are breast, ovarian, lung, cervical, colorectal, gastric,pancreatic, bladder, endometrial, kidney, and esophageal cancers, andpremalignancies and carcinoma in-situ such as cervical dysplasia,cervical intraepithelial neoplasia (CIN), bronchial dysplasia, atypicalhyperplasia of the breast, ductal carcinoma in-situ, colorectal adenoma,atypical endometrial hyperplasia, and Barrett's esophagus, and furthergestational trophoblastic cancers and gestational trophoblastic diseasein woman with a recent antecedent pregnancy.

In certain preferred embodiments of the methods of the invention, 5T4RNA or cDNA derived therefrom is amplified in a quantitative manner,thereby enabling comparison of amounts of 5T4 RNA present in a bodilyfluid such as blood plasma or serum from a human. In these embodiments,the amount of extracellular 5T4 RNA detected in an individual iscompared with a range of amounts of extracellular 5T4 RNA detected insaid bodily fluid in populations of humans known to have a premalignantor malignant disease, or known to be free from a premalignant ormalignant disease.

In alternative embodiments, said quantitative detection of 5T4 RNA inbodily fluids in an animal, most preferably a human, is provided forcomparing the amount of 5T4 RNA in said bodily fluid in pregnantindividuals that have a normal placenta, or that have placentaldisorders and conditions, particularly disorders and conditionscharacterized by or associated with trophoblast tissue includingplacental insufficiency, preeclampsia, eclampsia, gestationaltrophoblastic diseases such as molar pregnancy, gestationaltrophoblastic neoplasia, and conditions associated with inappropriatelyretained trophoblastic tissue, such as following labor and delivery, andfollowing recognized or unrecognized early terminations of thepregnancy, such as in spontaneous or incomplete abortions or followingmedical termination of the pregnancy.

The methods of the invention further provide methods for identifyingindividuals having a 5T4 expressing malignancy or premalignancy, therebypermitting rational, informed treatment options to be used for makingtherapeutic decisions. In particular, the methods of the invention areuseful in identifying individuals having a premalignancy or malignancythat would benefit from a 5T4-directed therapy, such as monoclonalantibody therapy, anti-sense therapy, and vaccine therapy.

Another advantageous aspect of the methods of the invention is as amarker for monitoring or assessing the adequacy of anticancer therapy,including surgical intervention, chemotherapy, biotherapy such asmonoclonal antibody therapy or vaccines, and radiation therapy, or fordetermining whether additional or more advanced therapy is required. Theinvention therefore provides methods for monitoring the response totreatment in such patients and for developing a prognosis in suchpatients.

The methods of the invention also allow identification or analysis of5T4 RNA, either qualitatively or quantitatively, in the blood or otherbodily fluid of an animal, most preferably a human that has completedtherapy, as an early indicator of relapsed cancer, impending relapse, ortreatment failure.

The invention provides methods for monitoring trophoblast RNA in plasmaor serum or other bodily fluids during a pregnancy. The methods of theinvention thereby permit placental monitoring in a pregnant woman,wherein quantitative evaluations of plasma or serum 5T4 RNA are mostpreferably made serially during a pregnancy, or wherein a singlequantitative evaluation of 5T4 RNA is compared to the normal serum orplasma 5T4 RNA values among females, most preferably women, havinghealthy pregnancies. The invention provides methods for comparing bloodlevels of RNA derived from the trophoblast to blood levels of RNAderived from the embryo and/or blood levels of RNA derived from maternalcells.

The methods of the invention also permit detection of retained placentaltissue in a woman following labor and delivery, or following earlytermination of a pregnancy such as in spontaneous or incompleteabortions (i.e., miscarriages).

Specific preferred embodiments of the present invention will becomeevident from the following more detailed description of certainpreferred embodiments and the claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention provides methods for detecting 5T4 RNA in bodily fluids inan animal, most preferably a human, and thereby enables detection ofcancerous or precancerous cells. The invention also provides methods fordetecting placental tissues that express 5T4, thereby permittingdetection of pregnancy and complications or conditions incident topregnancy.

In preferred embodiments of the methods of the invention, extracellularRNA particularly comprising 5T4 RNA is extracted from a bodily fluid.This extracted RNA is then amplified, either after conversion into cDNAor directly, using in vitro amplification methods in either aqualitative or quantitative manner, and using oligonucleotide primersspecific for 5T4 RNA or cDNA. The amplified product is then detected ineither a qualitative or a quantitative manner.

In the practice of the methods of the invention, 5T4 RNA may beextracted from a bodily fluid, including but not limited to whole blood,plasma, serum, urine, effusions, ascitic fluid, saliva, cerebrospinalfluid, cervical secretions, vaginal secretions, endometrial secretions,amniotic fluid, gastrointestinal secretions, bronchial secretionsincluding sputum, breast fluid or secretions or washings. Extraction canbe performed using, for example, extraction methods described inco-owned and co-pending U.S. patent application Ser. No. 09/155,152, theentire disclosure of which is hereby incorporated by reference. In apreferred embodiment, the bodily fluid is either blood plasma or serum.It is preferred, but not required, that blood be processed soon afterdrawing, and preferably within three hours, to minimize any degradationin the sample. In a preferred embodiment, blood is first collected byvenipuncture and kept on ice until use. Preferably within 30 minutes ofdrawing the blood, serum is separated by centrifugation, for example at1100×g for 10 minutes at 4 degrees centrigrade. When using plasma, bloodshould not be permitted to coagulate prior to separation of the cellularand acellular blood components. Serum or plasma can be frozen until use,for example at −70 degrees centigrade after separation from the cellularportion of blood. When using frozen blood plasma or serum, the frozenplasma or serum is rapidly thawed, for example in a water bath at 37degrees centigrade, and RNA is extracted therefrom with out undue delay,most preferably using a commercially available kit (for example thePerfect RNA Total RNA Isolation Kit obtained from Five Prime-ThreePrime, Inc., Boulder, Colo.), according to the manufacturer'sinstructions. Other alternative and equivalent methods of RNA extractionare further provided in co-owned and U.S. patent application Ser. No.09/155,152, now U.S. Pat. No. 6,329,179, incorporated herein byreference in its entirety.

Following extraction of RNA from a bodily fluid that contains 5T4 mRNA,the 5T4 mRNA or cDNA derived therefrom is amplified in vitro. Applicableamplification assays are detailed in co-owned and U.S. patentapplication Ser. No. 09/155,152, now U.S. Pat. No. 6,329,179, as hereinincorporated by reference, and include but are not limited to reversetranscriptase polymerase chain reaction (RT-PCR), ligase chain reaction,DNA signal amplification, amplifiable RNA reporters, Q-beta replication,transcription-based amplification, boomerang DNA amplification, stranddisplacement activation, cycling probe technology, isothermal nucleicacid sequence based amplification, and other self-sustained sequencereplication assays.

In preferred embodiments of the methods of the invention, 5T4 m RNA isconverted into cDNA using reverse transcriptase prior to in vitroamplification using methods known in the art. For example, a sample suchas 10 microliters (microL) extracted serum RNA is reverse-transcribed ina 30 microL volume containing 200 Units of Moloney murine leukemia virus(MMLV) reverse transcriptase (Promega, Madison, Wis.), a reaction buffersupplied by the manufacturer, 1 mM each dNTPs, 0.5 micrograms randomhexamer oligonucleotide primers, and 25 Units of RNAsin (Promega,Madison, Wis.). Reverse transcription is typically performed under anoverlaid mineral oil layer to inhibit evaporation, and incubated at roomtemperature for 10 minutes followed by incubation at 37 degrees C. forone hour.

Amplification oligonucleotide primers are selected to be specific foramplifying 5T4 nucleic acid. In a preferred embodiment, amplification isperformed by RT-PCR. In this embodiment, preferred oligonucleotideprimers have a nucleotide sequence as follows:

Primer 5T4-1: TCTTCGCCTCTTGTTGGC (gene location exon 2, 5T4 gene;Genbank accession #HSA012159; SEQ ID No. 1) Primer 5T4-2:TGCAGGAAGGAACGGGA (gene location exon 1, 5T4 gene; Genbank accession#HSA012159; SEQ ID No. 2) Primer 5T4-3: TTGGTAGGGAAGGAATTGGG (genelocation exon 1, 5T4 gene; Genbank accession #HSA012159; SEQ ID No. 3)Primer 5T4-1 and Primer 5T4-2 are particularly useful because they spanthe first intron and are therefore specific for cDNA and will notamplify contaminating genomic DNA (if any is present).

In one example of a preferred embodiment, 5T4 RNA is harvested fromserum or plasma, and RNA extracted therefrom by the Perfect RNA TotalRNA Isolation Kit (Five Prime—Three Prime, Inc., Boulder, Colo.)according to manufacturer's instructions. A sample of this extracted RNApreparation is reverse transcribed to cDNA, and RT-PCR for the 5T4 cDNAperformed using the primers described above. The cDNA is amplified in asingle-stage reaction in a thermocycler under a temperature profile thatproduces the specific fragment; typically, such a thermocycling profileconsists of an initial 10 minute incubation at 95 degrees C., followedby about 45 cycles of denaturation at 94 degrees C. for 30 seconds,annealing at 57 degrees C. for 30 seconds, and extension at 72 degreesC. for 30 seconds, followed by a final incubation at 72 degrees C. for 8minutes. Detection of the amplified product is achieved, for example bygel electrophoresis through a 5% Tris-borate-EDTA (TBE) agarose gel,using ethidium bromide staining for visualization and identification ofthe product fragment. The expected size of the product fragment is 101base pairs in length.

In a particularly preferred embodiment, 5T4 cDNA is amplified by RT-PCRin a hemi-nested, two stage amplification reaction. In this embodiment,the reaction mixture and amplification in the first stage are identicalto that of the single stage RT-PCR reaction described above, except thatthe reaction mixture for the first stage utilizes only 1 picomole eachof above-identified Primer 5T4-1 and Primer 5T4-2, and thermocyclingduring the first stage is performed using an otherwise identicaltemperature profile to the single stage method but for only 25 cycles.Following the first stage amplification, one-tenth volume of eachreaction mixture is transferred to fresh tubes, and a new reactionmixture is prepared that is identical to the mixture described aboveexcept that 10 picomoles each of Primer 5T4-1 and Primer 5T4-3 describedabove are utilized. The reaction mixture is then reamplified in athermocycler for 35 additional cycles under the above-describedtemperature profile. Detection of the amplified product fragment isachieved as described above, with the amplified product fragment being73 base pairs long.

In alternative preferred embodiments, amplified products can be detectedusing other methods, including but not limited to other gelelectrophoresis methods; capillary electrophoresis; ELISA ormodifications thereof, such as amplification using biotinylated orotherwise modified primers; nucleic acid hybridization using specific,detectably-labeled probes, such as fluorescent-, radioisotope-, orchromogenically-labeled probes; Southern blot analysis; Northern blotanalysis; electrochemiluminescence; laser-induced fluorescence; reversedot blot detection; and high-performance liquid chromatography.Furthermore, PCR product fragment detection may be performed in either aqualitative or quantitative fashion.

PCR product fragments produced using the methods of the invention can befurther cloned into recombinant DNA replication vectors using standardtechniques. RNA can be produced from cloned PCR products, and in someinstances the RNA expressed thereby, for example, using the TnT QuickCoupled Transcription/Translation kit (Promega, Madison, Wis.) asdirected by the manufacturer.

In another embodiment, restriction digestion may be performed upon thesingle-stage RT-PCR product with BamHI yielding two fragments ofapproximately 67 and 34 bp. The products of the restriction digestioncan be further amplified in a second stage amplification reaction usingappropriate primers.

In a preferred embodiment, 5T4 mRNA is amplified in a quantitativefashion thereby enabling comparison of the amount of extracellular 5T4mRNA in an individual's bodily fluid with the range of amounts of 5T4mRNA present in the bodily fluids of populations with cancer,premalignancy, pregnancy, recent pregnancy, or non-pregnant normalpopulations.

The methods of the invention as described above is not limited to bloodplasma or serum, and can be performed in like manner for detecting 5T4mRNA from other bodily fluids, including but not limited to whole blood,urine, effusions, ascitic fluid, saliva, cerebrospinal fluid, cervicalsecretions, vaginal secretions, endometrial secretions, amniotic fluid,gastrointestinal secretions, breast fluid or secretions, and bronchialsecretions including sputum. Although fractionation of the bodily fluidinto its cellular and non-cellular components is not required for thepractice of the invention, the non-cellular fraction may be separated,for example, by centrifugation or filtration of the bodily fluid.

The methods of the invention are useful in the practice of diagnosticmethods for detecting 5T4 mRNA in an animal, most preferably a human atrisk for developing or who has developed a premalignant or malignantneoplastic disease comprising cells expressing 5T4 mRNA. The inventionfurther provides a method of identifying animals, particularly humanswho are at risk for developing, or who have developed premalignancies orcancer of epithelial tissues and components of tissues, including butnot limited to breast, ovarian, lung, cervical, colorectal, gastric,pancreatic, bladder, endometrial, kidney, and esophageal cancers, aswell as premalignancies and carcinoma in-situ including but not limitedto cervical dysplasia and cervical intraepithelial neoplasia (CIN),bronchial dysplasia, atypical hyperplasia of the breast, ductalcarcinoma in-situ, colorectal adenoma, atypical endometrial hyperplasia,and Barrett's esophagus.

The diagnostic methods of the invention can be advantageously performedusing a diagnostic kit as provided by the invention, wherein the kitincludes oligonucleotide primers specific for 5T4 cDNA synthesis or invitro amplification or both, and/or specific probes, most preferablyoligonucleotide probes for detecting 5T4 RNA or cDNA or in vitroamplified DNA fragments thereof. The kit may further include methods andreagents for extracting 5T4 RNA from a bodily fluid, wherein the bodilyfluid is most preferably but not limited to blood plasma or serum.

The inventive methods have significant advantages in assigning andmonitoring therapies not specifically directed at 5T4 expressing cells,including chemotherapy, radiation therapy, and surgery, as well as5T4-specific or directed therapies such as 5T4-directed monoclonalantibody therapy and 5T4-directed vaccine therapy. The methods of theinvention permit stratification and selection of individuals,particularly individual human patients likely to benefit from5T4-specific or directed therapy. The inventive methods are also usefulfor monitoring response, relapse, and prognosis of 5T4-producingneoplastic disease. Of particular value, the invention allows adetermination that a 5T4-directed therapy is therapeutically indicatedeven in cases of premalignancy, early cancer, or occult cancers orminimum residual disease, as well as when metastatic disease is present.Thus, the invention permits therapeutic intervention when tumor burdenis low, immunologic function is relatively intact, and the patient isnot compromised, all increasing the potential for cure.

The methods of the invention further enable 5T4 RNA to be evaluated inblood plasma, serum or other bodily fluid in combination with detectionof other tumor-associated or tumor-derived RNA or DNA in a concurrent orsequential fashion, such as in a multiplexed assay or in a chip-basedassay, thereby increasing the sensitivity or efficacy of the assay inthe detection or monitoring of neoplastic disease. For example, but notas a limitation, 5T4 RNA can be detected in blood, plasma, serum, orother bodily fluid in combination with detection oftelomerase-associated RNA such as hTR and/or hTERT, or in combinationwith detection of human papillomavirus DNA.

The invention also provides methods for detecting 5T4 RNA in pregnantanimals, most preferably pregnant women, and thereby provides a methodof monitoring the competency and health of the placenta. The amount of5T4 RNA present in blood plasma or serum of a pregnant woman generallycorresponds to the amount of placental tissue present, the health of theplacenta, and duration of pregnancy. Quantitative serial determinationsof 5T4 RNA in blood plasma and serum thereby enable non-invasiveassessment and monitoring of the placenta during pregnancy. Comparisonof quantitative values of plasma or serum 5T4 RNA (or othertrophoblastic RNA) within a particular pregnant woman to a normal rangewithin healthy pregnancies at any given stage of pregnancy, or incomparison to embryonic development, can be utilized to indicateplacental pathology, such as may occur in preeclampsia, eclampsia, andplacental insufficiency, and the appropriateness of medicalintervention.

While the placenta is normally delivered completely following labor, insome cases the placenta or portions thereof may be retained withpotential deleterious consequences to the woman. Trophoblastic tissuemay be inappropriately retained post-partum, such as in association withplacental accreta and placental increta, or following antecedentpregnancies, such as pregnancies characterized by blighted ovumsyndrome, spontaneous or incomplete abortions (miscarriages) withretained products of conception, or following medical terminations ofthe pregnancy. Furthermore, trophoblastic tissue persists in gestationaltrophoblastic diseases, including molar pregnancy, and gestationaltrophoblastic neoplasia. Thus, the methods of the invention can be usedto determine the presence of retained placental tissue or products ofconception. In these uses of the inventive methods, persistence oftrophoblastic RNA in plasma or serum in a woman with a recent antecedentpregnancy is indicative of retained trophoblastic tissue. The inventionfurther permits detection, evaluation, and monitoring of gestationaltrophoblastic diseases and gestational trophoblastic neoplasia thereby.

The invention also provides methods for detecting trophoblastic tissuein a pregnant or recently pregnant woman, wherein said method comprisesextracting trophoblast RNA from the blood plasma or serum of said woman,wherein the trophoblast RNA is not normally detectable at similarquantitative or qualitative levels in the blood of a healthynon-pregnant woman. It will be understood that the methods of theinvention as applied to the pregnant or recently pregnant woman fordetecting 5T4 RNA are applicable to any RNA that is primarily oftrophoblastic origin and which is not normally detectable in blood ofhealthy non-pregnant woman; this application of the inventive methodsrequires that the nucleic acid sequence of the RNA is known to the art,permitting production of oligonucleotide primers therefore. In apreferred embodiment of the methods of the invention the trophoblast RNAspecies is 5T4 RNA. In other preferred embodiments of the inventivemethods the trophoblast RNA species is, but is not limited to,beta-human chorionic gonadotrophin (hCG) RNA, alpha-hCG RNA,carcinoembryonic RNA (CEA RNA), placental alkaline phosphatase RNA (PLAPRNA), and pregnancy-specific glycoprotein RNA (PSG RNA). Furthermore,multiple trophoblast RNA species may be evaluated from plasma or serumin a concurrent or sequential fashion, such as in a multiplexed assay orin a chip-based assay.

The methods of the invention and preferred uses for the methods of theinvention are more fully illustrated in the following Examples. TheseExamples illustrate certain aspects of the above-described method andadvantageous results. These Examples are shown by way of illustrationand not by way of limitation.

Example 1 Detection of 5T4 mRNA in Placental and Carcinoma Tissue

5T4 mRNA was detected in placental tissue and other normal and canceroushuman tissues as follows. Normal placenta was obtained within hours ofdelivery and stored at −80 degrees C. until use. Normal human tissuesfrom other organs, consisting of normal tissue from brain, kidney,liver, skeletal muscle, spleen, and myocardium, were obtained fromautopsies within 12 hours of death, snap frozen, and stored at −80degrees C. until use. In addition, eight human breast cancer specimensand sixteen human lung cancer specimens were available asformalin-fixed, paraffin-embedded tissue obtained at times of biopsy orsurgery.

Placental RNA and RNA from normal tissues of the brain, kidney, liver,skeletal muscle, spleen and myocardium were extracted from five 20micron slices of each fresh tissue sample using the Perfect RNA TotalRNA Isolation kit (Five Prime—Three Prime, Inc., Boulder Colo.),according to manufacturer's instructions. For each tissue specimen,extracted RNA was then reverse transcribed to cDNA in a 30 microL volumecontaining 200 Units of Moloney murine leukemia virus (MMLV) reversetranscriptase (Promega, Madison, Wis.), a reaction buffer supplied bythe manufacturer, 1 mM each dNTPs, 0.5 micrograms random hexameroligonucleotide primers, and 25 Units of RNAsin (Promega, Madison,Wis.). Reverse transcription was performed under an overlaid mineral oillayer to inhibit evaporation, and incubated at room temperature for 10minutes followed by incubation at 37 degrees C. for one hour. RT-PCR wasthen performed as either a single-stage amplification or as a two-stage,hemi-nested amplification, wherein for the single-stage amplification, 5microliters of the 5T4 cDNA was used in a final volume of 50 microL in areaction mixture containing 1 U of Amplitaq Gold (Perkin Elmer Corp.,Foster City, Calif.), 1× reaction buffer supplied with Amplitaq by themanufacturer, 1.5 mM MgCl₂, 200 microM each dNTP, and 10 picomoles eachof Primer 5T4-1 and Primer 5T4-2 identified above. The mixture wasamplified in a single-stage reaction in a thermocycler under atemperature profile consisting of an initial 10 minute incubation at 95degrees C., followed by 45 cycles of denaturation at 94 degrees C. for30 seconds, annealing at 57 degrees C. for 30 seconds, and extension at72 degrees C. for 30 seconds, followed by a final incubation at 72degrees C. for 8 minutes. The amplified product (101 bp) was detected bygel electrophoresis through a 5% agarose gel with staining of productsby ethidium bromide for identification of the product. From theplacental tissue specimens, a 5T4-specific RT-PCR product was produced,indicating that 5T4 mRNA was present in the placenta. For the two-stage,hemi-nested RT-PCR amplification reactions, the reaction mixture andamplification in the first stage were identical to that of thesingle-stage RT-PCR reaction described, except that only 1 picomole eachof Primer 5T4-1 (SEQ ID No. 1) and Primer 5T4-2 (SEQ ID No. 2) wereused, and thermocycling was performed for only 25 cycles. Following thefirst stage amplification, one-tenth of the reaction mixture wastransferred to fresh tubes, and a new reaction mixture was prepared thatwas the same as the previous mixture except for the primers, wherein 10picomoles each of Primer 5T4-1 (SEQ ID No. 1) and Primer 5T4-3 (SEQ DNo. 3) as previously described were used. The reaction mixture was thenreamplified for 35 additional cycles at the above temperature profile.The 5T4 amplified product fragment from placenta was detected by gelelectrophoresis through a 5% agarose gel, with staining of products byethidium bromide for identification of the product, with the expectedamplified product being 73 base pairs long. In comparison, of allsamples of normal tissue from which RNA was prepared, none of theautopsy tissues contained amplifiable 5T4 mRNA. As a positive control,c-abl mRNA in these tissues could be demonstrated by PCR amplificationusing c-abl specific primers.

5T4 mRNA was detected in human tumor samples as follows. Formalin-fixedcancer tissues were used, with RNA prepared from two 10 micron slices ofeach paraffin-embedded lung and breast cancer specimen, processedaccording to the method of Bianchi et al. (1991, Am. J. Path. 138:279-284), with the exception that harvested RNA was used directly (5 or18 microliters of 500 microliters total after organic extractions) forreverse transcription rather than following ethanol precipitation. Theextracted RNA was then amplified by RT-PCR, using the protocol describedabove using either a single-stage or two-stage PCR assay. Amplifiedproducts were detected by gel electrophoresis through a 5% agarose geland visualized by staining with ethidium bromide for identification ofthe products. 5T4 mRNA amplified products were detectable by gelelectrophoresis in 3 lung cancer specimens, and in 3 breast cancerspecimens, indicating the presence of 5T4 mRNA in these specimens.

Example 2 Detection of 5T4 mRNA in Serum from Cancer Patients

Sera were prepared from the blood of 5 patients with breast cancer and14 patients with lung cancer in the manner described above. Blood waskept on ice following venipuncture and serum was separated within 30 minof blood draw by centrifugation at 1100×g for 10 minutes at 4 degreesC., and then the serum was stored frozen at −70 degrees C. untilassayed. At the time of assay, the sera were rapidly thawed by placingserum samples in a water bath heated to 37 degrees C. RNA was thenextracted from 1.75 mL of sera using the Perfect RNA Total RNA IsolationKit (Five Prime-Three Prime, Inc., Boulder, Colo.) according to themanufacturer's instructions. Ten microL of the extracted RNA was thenreverse transcribed using MMLV reverse transcriptase (Promega, Madison,Wis.) in the manner described above, wherein 10 microL of extractedserum RNA was reverse-transcribed in a 30 microL volume containing 200Units of MMLV reverse transcriptase (Promega), a reaction buffersupplied by the manufacturer, 1 mM each dNTPs, 0.5 micrograms randomhexamer mixture of oligonucleotide primers, and 25 Units of RNAsin(Promega), with incubation at room temperature for 10 minutes followedby incubation at 37 degrees C. for one hour. The 5T4 cDNA was thenamplified by PCR using the primers and amplification parameters asdescribed in Example 1 either by single stage or two stage, hemi-nestedRT-PCR amplification. All specimens were evaluated by the single stagePCR amplification, and then separately evaluated using the moresensitive two-stage, hemi-nested PCR amplification reaction. For thesingle-stage amplification, 5 microliters of the 5T4 cDNA was used in afinal volume of 50 microL in a reaction mixture containing 1 U ofAmplitaq Gold (Perkin Elmer Corp., Foster City, Calif.), 1× reactionbuffer, 1.5 mM MgCl₂, 200 microM each dNTPs, and 10 picomoles each ofPrimer 5T4-1 (SEQ ID No. 1) and Primer 5T4-2 (SEQ ID No 2). The mixturewas amplified in a single-stage reaction in a thermocycler under atemperature profile consisting of an initial 10 minute incubation at 95degrees C., followed by 45 cycles of denaturation at 94 degrees C. for30 seconds, annealing at 57 degrees C. for 30 seconds, and extension at72 degrees C. for 30 seconds, followed by a final incubation at 72degrees C. for 8 minutes. The amplified product was detected by gelelectrophoresis through a 5% agarose gel with staining of amplifiedproducts by ethidium bromide for identification of the product. For thetwo-stage, hemi-nested RT-PCR amplification reactions, the reactionmixture and amplification in the first stage were identical to that ofthe single-stage RT-PCR reaction described above, except that only 1picomole of Primer 5T4-1 (SEQ ID No. 1) and Primer 5T4-2 (SEQ ID No. 2)were used, and thermocycling was performed for only 25 cycles. Followingthe first stage amplification, one-tenth of the reaction mixture wastransferred to fresh tubes, and a new reaction was prepared that was thesame as the previous mixture except for the primers, wherein 10picomoles each of Primer 5T4-1 (SEQ ID No. 1) and Primer 5T4-3 (SEQ IDNo. 3) were used. The reaction mixture was then reamplified for 35additional cycles at the above temperature profile. The 5T4 amplifiedproduct was detected by gel electrophoresis through a 5% agarose gel,with staining of products by ethidium bromide for identification of theproduct, with the expected amplified product being 73 base pairs inlength. Two of the 19 patients, both with lung cancer, had sera positivefor 5T4 mRNA using the single-stage PCR assay, while the more sensitivehemi-nested two stage PCR assay demonstrated sera to be positive for 5T4mRNA in 8 patients, including those of 2 breast cancer patients and 6lung cancer patients (including both patients positive with thesingle-stage assay). Positive and negative controls consisting ofplacental tissue RNA as a positive control and a cDNA absent blank as anegative control were appropriate for all reactions.

Example 3 Detection of 5T4 RNA in the Plasma of a Woman Postpartum

A 27 year old woman delivers a healthy baby at term following a normallabor and delivery. Thirty-six hours following delivery (postpartum) thewoman develops a fever, raising the clinical question of possibleretained placental tissue. Plasma is drawn from the woman, and 5T4 RNAis demonstrated in her plasma at higher than expected levels using themethods as outlined in the invention. The finding of 5T4 RNA in theplasma supports the clinical suspicion of retained placental tissue, andthe woman undergoes further surgical procedures.

Example 4 Detection of 5T4 RNA in the Serum of a Woman Following aSpontaneous Loss of Pregnancy

A 22 year old woman in her first pregnancy suffers a spontaneousabortion (miscarriage) following 7 weeks of pregnancy. She reports heavybleeding and passing tissues of conception, but it is unclear whetherall fetal tissue has passed. Three days following the event, blood isdrawn from the woman and the serum tested for the presence of 5T4 RNAusing the methods of the invention. The presence of 5T4 RNA is confirmedin the woman, suggesting the presence of retained trophoblastic tissue.The woman consequently undergoes a further surgical procedure to removethe remaining tissues of conception.

Example 5 Detection of 5T4 RNA in the Serum of a Woman with GestationalTrophoblastic Disease/Neoplasia Following an Antecedent Pregnancy

A 25 year old woman suffers a first trimester miscarriage. Four monthsfollowing her miscarriage, serum is obtained and the methods of theinvention used to demonstrate that the serum contains high levels of 5T4RNA. A diagnosis of gestational trophoblastic disease is made and thewoman treated surgically. Serum 5T4 RNA levels are quantitativelyfollowed with a transient decline in levels. Six months following hersurgical procedure, serum 5T4 RNA levels are noted to be persistentlyrising, and a chest x-ray demonstrates a new pulmonary lesion.Gestational trophoblastic neoplasia is suspected based upon elevated 5T4RNA and elevated serum beta-HCG RNA levels, and the diagnosis isconfirmed by biopsy. The patient subsequently is treated by chemotherapyincluding a 5T4-directed therapy.

Example 6 Use of Plasma 5T4 mRNA in the Monitoring of Cancer

A 42 year old man is diagnosed with renal cancer. Plasma 5T4 mRNA isquantitatively measured using the methods of the invention prior toinitiation of therapy. The man then undergoes surgical resection of hisrenal cancer. The plasma 5T4 mRNA levels are then followed using themethods of the invention in a serial manner to aid in the determinationof prognosis, relapse, and recurrence, as well as response to subsequenttherapies.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims.

1. A method of detecting 5T4 RNA or beta-human chorionic gonadotropin(beta-HCG) RNA in blood plasma or serum from a woman with a gestationaltrophoblastic neoplasia that is said 5T4 RNA or beta-HCG RNA-expressingneoplasia comprising the steps of: a) extracting extracellular RNA fromblood plasma or serum from a woman having a gestational trophoblasticneoplasia that is 5T4 RNA or beta-HCG RNA-expressing neoplasia, whereina portion of the extracellular RNA comprises said 5T4 RNA or beta-HCGRNA; b) amplifying a portion of the extracellular RNA or cDNA preparedtherefrom and producing an amplified product, wherein the amplificationis performed in either a qualitative or quantitative fashion usingoligonucleotide primers specific for said 5T4 RNA or cDNA producedtherefrom, or for said beta-HCG RNA or cDNA produced therefrom; and c)detecting said 5T4 RNA in the blood plasma or serum from said woman bydetecting the amplified product when the amplification is performedusing said oligonucleotide primers specific for said 5T4 RNA or cDNAproduced therefrom, or detecting said beta-HCG RNA in the blood plasmaor serum from said woman by detecting the amplified product when theamplification is performed using said oligonucleotide primers specificfor said beta-HCG RNA or cDNA produced therefrom.